LOCUS       sequence-476345-        5330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of high affinity green fluorescent pyruvate 
            biosensor GreenPy1High.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5330)
  AUTHORS   Imai S, Hario S, Suerte C, Yamaguchi I, Sakoi K, Terai T, 
            Takahashi-Yamashiro K, Campbell RE
  TITLE     High-performance genetically-encoded green and red fluorescent 
            biosensors for pyruvate
  JOURNAL   bioRxiv 2025.04.17.649293
REFERENCE   2  (bases 1 to 5330)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.04.17.649293"
COMMENT     Sequence Label: pBAD-GreenPy1High
FEATURES             Location/Qualifiers
     source          1..5330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..47
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    62..202
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(128..147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     288..310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(466..1344)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1371..1655
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1578..1597
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     CDS             1701..1718
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1722..1754
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(1758..1766,1767..1781)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     2345..2366
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2472..2493)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2733..2752)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(3101..3118)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3271..3357
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3449..3476
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3495..3586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3587..3655,3656..4447)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3805..3824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4618..5206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5107..5126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
       61 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
      121 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
      181 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
      241 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
      301 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagcagatca attcgcgcgc
      361 gaaggcgaag cggcatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac
      421 cctatgctac tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac
      481 ggctacatca ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt
      541 gcatttttta aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac
      601 ggtggcgata ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc
      661 ctcgcgccag cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga
      721 cggcgacaag caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg
      781 atcgctgatg tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc
      841 gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc
      901 cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg
      961 cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt
     1021 aagccattca tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg
     1081 agcctccgga tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc
     1141 cggtcggcaa acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag
     1201 attgagaata taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt
     1261 ggcctcaatc ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag
     1321 gggatcattt tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa
     1381 ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac
     1441 caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac
     1501 aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca
     1561 cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg
     1621 acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc taacaggagg
     1681 aattaaccat ggggggttct catcatcatc atcatcatgg tatggctagc atgactggtg
     1741 gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatccgagc tcgagaatgg
     1801 tgcaatcttt ttccgatccg ctggtagaac tgcttccaga tcacccggaa tcccaatatg
     1861 acttgctcga agcgcgccat gcgctggaag ggatcgcagc atattacgct gccttgcgca
     1921 gcacagatga agataaagag aggatcagag agttgcatca tgcgattggg cttgcccaac
     1981 aaagtggtga tctcgatgcc gaaagtaacg ctgtacttca gtatcaaacc gcggtgaccg
     2041 aggcagcgca caatgtggtc cttttgcact tgttgagatg tatggagcct atgctcgcac
     2101 agaatatccg acaaaacttt gagctgctgt accgtcataa cgtctatgtc atggccgaca
     2161 agcagaggaa cggcatcaag gcgaacttca agatccgcca caacatcgag ggcggcggcg
     2221 tgcagctcgc ctaccaccac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2281 ccgacaacca ctacctgtgc acccagtcca aactttcgaa agaccccaac gagaagcgcg
     2341 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2401 tgtacatggg cggtaccgga gggagcatgg tgagcaaggg cgaggagctg ttcaccgggg
     2461 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2521 gcgagggtga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2581 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2641 tcggccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2701 gctacatcca ggagcgcacc atcttcttcg aggacgacgg caactacaag acccgcgccg
     2761 aggtgaagtt cgagggcgac accctggtga gccgcatcga gctgaagggc atcgacttca
     2821 aggaggacgg caacatcctg gggcataagc tggagtacaa ctcgtggcga cgagagatgc
     2881 tgccgctcgt gtcttcccat cgcactcgca tatttgaagc tattatggca ggaaaaccgg
     2941 aggaagccag ggaagcgtcc cataaacact tggcctttat tgaggaaatc ctgctggacc
     3001 ggtcaaggga ggaatcacgc cgcgaacgct cattgagacg gctggagcaa agaaagaatt
     3061 gaaagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag
     3121 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
     3181 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
     3241 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     3301 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     3361 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     3421 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     3481 tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     3841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     4321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaacaaccac cgctaccagc
     4681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4741 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
//
